PREEMERGENCE WEED CONTROL USING NATURAL HERBICIDES

Abstract: A method of selectively inhibiting growth of unwanted plants in a plot of soil is provided comprising the application of a plant protein hydrolysate to the plot of soil, prior to emergence of the unwanted plants and at a concentration of application which inhibits the growth of the unwanted plants. The invention further provides a composition useful as a selective preemergence herbicide comprising an effective amount of a peptide selected from the group consisting of Gln-Gln, Ala-Asn, Ala-Gln, Gly-Ala, Ala-Ala, Leu-Ser-Pro-Ala-Gln and mixtures thereof.

Description: PREF IERGENCE WEED CONTROL USING NATURAL HERBICIDES BACKGROUND OF THE INVENTION Herbicides have been widely employed to destroy unwanted plants or "weeds", to prevent their growth on bare ground or in established crops, and to promote the growth of desirable plants, such as grains, fruits, and vegetables. In fact, millions of pounds of herbicide are applied directly to the soil on an annual basis. In general, herbicides consist of two types, non- selective and selective. Non-selective herbicides kill all plant life on the plot of soil on which they are applied. Selective herbicides, on the other hand, kill or inhibit the establishment of certain types of plant life, such as weeds, while leaving the desirable, surrounding crops on which they are applied relatively undamaged. Examples of selective herbicides include phenolics, carbamates, and dinitroanilines. One way to selectively eliminate unwanted plants without injuring surrounding plant life is to inhibit germination or establishment of the seeds of the unwanted plants. In order to accomplish this, a herbicide must be applied before the unwanted plants emerge from the soil, either to a bare plot of soil into which established plants will be transplanted, or to a plot of soil comprising an established stand of desirable plants, but relatively few weeds. Such herbicides are often referred to as preemergence herbicides. While various types of herbicides exist, most of them are based on synthetic chemical toxins. As a result of their toxic nature, they are undesirable for many applications. This is particularly a problem when these materials come in contact with the public as is the case in turfgrass areas and in the production of food crops consumed by humans. While synthetic chemical herbicides may effectively destroy unwanted plant life, they may contaminate the soil and the crops themselves. They may also contaminate the ground water as a result of run off or erosion. The disadvantages of synthetic chemical herbicides have become more visible as a result of heightened public awareness and concern for environmental protection and consumer safety. This in turn has led to the search for non-toxic, natural herbicides which can provide a greater margin of safety for the public and for the environment. In the area of herbicides or insecticides, however, few effective materials derived from naturally-occurring sources are known. Bacillus thurigiensis (Bt toxins), Bacillus popilliae, Serratia eritomophila, Puccinia chonάillina, and Sclewtinia sclerotiotwn represent some examples of natural herbicides and insecticides that currently exist. Corn gluten meal is capable of inhibiting root growth of germinating plants, while no damage is observed to plants that have formed a mature root system. Christians (U.S. Pat. No. 5,030,268) discloses that this material is useful as a natural preemergence herbicide for various plant production systems, including turfgrass areas, where it acts to inhibit the establishment of annual weeds, such as crabgrass (Digitaria spp.). Corn gluten meal, however, is essentially water-insoluble. This characteristic limits its use as e an herbicide for some applications. Since corn gluten meal is insoluble and cannot be dissolved and sprayed, it is difficult to apply evenly. As a result, there is a risk that the soil on which it is applied will not be completely covered, thereby significantly reducing its effectiveness. Also, sprayable herbicides are advantageous for application to certain crops. The effectiveness of a herbicide also depends upon its ability to permeate the soil. Water-insoluble or slightly soluble materials do not permeate the soil as well as do water-soluble materials. Factors such as wind or drought can further reduce the availability of such materials. Therefore, a continuing need exists for potent, natural preemergence herbicides which are also highly water dispersible and/or water soluble. SUMMARY OF THE INVENTION This invention provides a selective, non-toxic preemergence herbicide for use on soil plots to control both broadleaf and grassy weeds. Plant protein hydrolysates, preferably selected from the group consisting of corn gluten hydrolysate, wheat gluten hydrolysate, soy protein hydrolysate and mixtures thereof, have been found to provide water-soluble materials which are at least as active as com gluten meal as preemergence herbicides. For example, dry corn gluten hydrolysate prepared as disclosed hereinbelow has been observed to completely stop root formation of test species at an application level of 0.24 g/dm 2 in controlled environmental chambers in the laboratory and has been observed to prevent plant establishment by 96% at a level of 1.72 in greenhouse trials on soil. The examples hereinbelow demonstrate that corn gluten hydrolysate, wheat gluten hydrolysate and soy protein hydrolysate can be used as a growth-regulating material to inhibit root formation of germinating weeds in agricultural end-use settings. These hydrolysates can also be used safely with both broadleaf and grassy crops, and thereby act as natural preemergence herbicides. The plant protein hydrolysates can be applied to a plot of soil prior to transplanting the desirable plants, or can be applied to a plot of soil which already has a stand of established desirable plants thereon. Because these materials are protein hydrolysates of natural plant materials, they should be useful products for use in agriculture as a substitute for synthetic herbicides. During studies designed to isolate and identify one or more active components of corn gluten, we unexpectedly found that hydrolyzed protein from corn gluten provided an effective water-soluble, preemergence herbicide that is much more active than the corn gluten meal itself. Furthermore, investigation of these hydrolysates led to the isolation and identification of five dipeptides and a pentapeptide which are highly effective as selective preemergence herbicides. Thus, the present invention also provides a herbicidal composition comprising an effective herbicidal amount, a peptide selected from the group consisting of glutøminyl-glutamine (Gln- Gin), alaninylasparagine (Ala-Asn), alanmyl-glutamine (Ala-Gin), glycinyl- alanine (Gly-Ala), alaninyl-alanine (Ala-Ala), leucinyl-serinyl-prolinyl- alaninyl-glutamine (Leu-Ser-Pro-Ala-Gln) and mixtures thereof, in combination with a compatible carrier vehicle. Compatible carrier vehicles are preferably non-toxic and include liquids such as water, used alone or in combination with non-toxic co-solvents and art-recognized surfactants, stabilizers, buffers and the like. Solid vehicles include the finely-dispersed carrier vehicles employed to deliver dust-type herbicides. When applied to a plot of soil, either prior to planting said plot with established desirable plants, or having an established stand of desirable plants thereon, the present hydrolysates, dipeptides, pentapeptide, or mixtures thereof inhibit the root formation of the germinating seeds of the undesirable plants or "weeds" and thus inhibit or completely block their growth and emergence. Following application of these materials to the soil and planting established desirable plants in said plot, additional amounts can be applied as needed, to prevent the growth of undesirable plants while not inhibiting the growth of the desirable plants, or otherwise harming them. The present dipeptides, the pentapeptide or mixtures thereof, are also effective to inhibit the emergence of a wide variety of weeds, both broadleaf and grassy, while not harming established desirable plants, both broadleaf (dicot) and grassy (monocot). Thus, the present dipeptides function as a nontoxic, selective, natural preemergence herbicides when applied at a wide variety of concentrations and intervals to the target site. As used herein, the term "plot of soil" is intended to broadly cover volumes of solid plant support material such as the mixture of organic and inorganic materials conventionally referred to as "soil," as well as synthetic soils (or "soiless soils") and homogenous solid supports such as beds of pebbles, sand, moss and the like. The solid plant support material may be potted, or otherwise contained, or may be a preselected portion of the ground. The present invention also provides a two-stage chromatographic method to isolate the present dipeptides which sequentially applies gel filtration and reverse-phase high pressure liquid chromatography (HPLC) to an aqueous solution of plant protein hydrolysate, i.e., com gluten hydrolysate. The stationary phase for gel filtration preferably has an exclusion limit of less than 1500 daltons, and consists of crosslinked dextran beads such as the Sephadex resins available from Sigma Chem. Co. The present dipeptides and the pentapeptide are identified using conventional three-letter amino acid abbreviations and are read from the amino terminus (left) to the carboxy terminus (right). Although the present invention has been exemplified primarily by reference to com, wheat and soy hydrolysates, due to the similarity in the amino acid compositions of the various plant proteins, it is believed that a wide variety of plant protein hydrolysates would be useful in the practice of the invention, including hydrolysates from other grains and legumes. DETAILED DESCRIPTION OF THE INVENTION Com gluten meal is commercially available as a byproduct of com milling. It is made by drying the liquid gluten stream separated from com during com wet milling processing. In the wet milling process of com, the following fractions are obtained: com starch, com oil, defatted com germ, com hulls, com steep liquor, and com gluten (the protein fraction). Com gluten is typically separated from the starch stream by centrifugation to yield a thick, yellow slurry of com gluten containing 15 to 20% solids. Conventionally, com gluten is filtered and dried to produce solid com gluten meal, which is sold as an animal feed product. Com gluten meal is quite insoluble in water and is typically composed of the materials listed in Table 1, below. TABLE I Com Gluten Meal Component %, Dry Basis Protein 60-70 Carbohydrate 20-25 Fat 3-5 Ash 3-5 A. Hydrolysates The present plant protein hydrolysates are preferably prepared by a process comprising treating an aqueous sluπy of a plant protein such as corn or wheat gluten or soy protein with acid or with one or more enzymes. Preferably, the plant protein is treated with one or more proteases, and most preferably, is pre-treated with one or more amylases. For example, the proteinaceous slurry may be treated with amylases, followed by filtration to remove the solubilized carbohydrates. The insoluble residue is then treated with one or more proteases to solubilize the protein components. After pH adjustment with acid, the slurry is filtered and/or centrifuged. The effluent is dried in a conventional manner to yield "com gluten hydrolysate," "wheat gluten hydrolysate," or "soy protein hydrolysate," which is essentially water soluble (>90% at 10 g/100 ml). Alternatively, the protein slurry can be treated with proteases alone and the entire reaction mixture dried, or the reaction mixture may be centrifuged or filtered and the supernatant or filtrate dried in an appropriate manner, to yield a soluble plant protein hydrolysate. To prepare com gluten hydrolysates, the liquid com gluten (15- 20% solids) is preferably diluted with water to a solids concentration of about 5 to 20%) and the pH adjusted to about 6.0 to 8.0, preferably to about pH 6.5. The appropriate amylase is added (0.1 to 1.0% dry basis (DB)) and the sluπy jet cooked at 280° to 340°F., preferably at 320°F. for 3-4 minutes. The cooked slurry is then adjusted to about pH 4 to 5, cooled to 140°F. and, optionally, a saccharifying amylase (glucoamylase) is added (0.01 to 0.1% DB) and the slurry maintained at 140°F. for 8-18 hours, preferably about 12 hours. The slurry is then filtered and washed and the filtrate and washings discarded. The filter cake is reslurried in water to 5 to 20% solids (preferably about 10%)) and adjusted to pH 7.5 to 9 with Ca(OH) 2 . An alkaline protease is then added (0.1%) to 1% DB) and the slurry is maintained at 50° to 60°C. for 2 to 6 hours, or until the pH remains constant. The slurry is then adjusted to pH 6.0 to 6.8 (preferably pH 6.2), the precipitated Ca 3 (P0 4 ) 2 and any insoluble residue is removed by filtration. The clear filtrate is then dried in an appropriate manner (i.e., spray drying, drum drying, etc.) to yield a dry solid product having greater than about 80-90%) protein (Kjeldahl nitrogen), and which is essentially water-soluble at 10 wt-% concentration. On a dry basis, the com gluten hydrolysate will have a nitrogen content of at least about 8%, i.e., about 8-11.2%), most preferably at least about 14.4%>. The dry product can be applied by the use of conventional spreaders or dusters used for solid fertilizers or herbicides and can be applied as a dust, pellets, granules and the like. Com gluten hydrolysate can be applied at a level of 0.003-10 g/dm 2 of soil area, preferably at a level of about 0.5-4 g/dm 2 of soil area. Com gluten meal hydrolysate may also be freely dissolved or suspended in water and thus can be readily applied by delivery systems employed for the application of liquid herbicides, such as by spraying and watering. The amount of aqueous plant protein hydrolysate which is applied can be varied over a wide range depending on soil type, field contour, target species and the like. In some cases, it is preferable to combine or mix the com gluten hydrolysate with the soil. B. Peptides The five dipeptides isolated from com gluten meal hydrolysate that have been demonstrated to inhibit root growth of plants at the time of germination are glutaminyl-glutamine (Gln-Gln), alaninyl-asparagine (Ala- Asn), alaninyl-glutamine (Ala-Gin), glycinyl-alanine (Gly-Ala), and alaninyl- alanine (Ala-Ala). Additionally, a pentapephole, leucinyl-serinyl-prolinyl- alaninyl-glutamine, (Leu-Ser-Pro-Ala-Gln), isolated from com gluten hydrolysate has also been demonstrated to exhibit root growth of plants at the time of germination. The present peptides were isolated by subjecting aqueous solutions of com gluten hydrolysate to column chromatography (gel filtration). Herbicidal fractions in the eluate were identified by their ability to inhibit the germination of seeds, such as seeds of grassy weeds, in an in vitro assay. The active fractions were further purified by reverse phase high performance liquid chromatography. The bioactive fractions were then derivatized, purified further by chromatography and the resultant peptides were sequenced to identify the peptidyl components. The bioactive peptides identified can be readily synthesized by methods known to the art. Peptides Ala-Ala, Ala-Asn, Ala-Gin and Gly-Ala are available from Sigma Chem. Co., St. Louis, Mo.; peptide Gln-Gln is available from Bachem Bioscience Inc. In use, one or more of the present peptides are combined with an effective amount of a carrier vehicle, i.e., at about 0.25-25 wt-%> of the vehicle, and applied to the target soil plot/crop by conventional means, such as spraying, watering, spreading, dusting and the like. Suitable vehicles include water or water-alcohol mixtures, optionally in combination with minor but effective amounts of surfactants, solubilization aids, stabilizers, buffers and the like. Solid carrier vehicles include those commonly employed to apply herbicides to target areas, such as ground com cobs, clay and the like. Preferred application rates for the herbicidal dipeptide, pentapeptide, or mixtures thereof are about 0.003-5 g/άm 2 , preferably about 0.25-3.0 g/dm 2 , of soil per application. The herbicidal composition can be simply surface-applied, or it can be mixed into the upper layer of the soil following application. The present peptides can also be used to augment the herbicidal activity of com gluten meal or plant protein hydrolysates. C Efficacy It is believed that liquid or solid plant protein hydrolysates and isolated peptides thereof will be effective to prevent the emergence of a wide variety of undesirable plants, including broadleaf weeds, such as smartweed, velvetleaf, redroot, pigweed, lambsquarters, latchweed bedstraw, black medic, buckhom plantain, annual purslane, black nightshade; and grassy weeds such as crabgrass, annual bluegrass, creeping bentgrass, barnyard grass, orchard grass, woolly cupgrass, foxtails, shattercane, Kentucky bluegrass, Bermuda grass, perennial ryegrass and tall fescue. Thus, com gluten hydrolysate, soy protein hydrolysate, or wheat gluten hydrolysate, the present peptides and mixtures thereof, can be used as preemergence herbicides for application to established desirable plants, including both monocotyledonous plants and dicotyledonous plants. Monocotyledonous crops include the grains; corn, sorghum, rice, oats, wheat, flax, rye, millet, turfgrasses and the like. Dicotyledonous crops include fruits, fibers, herbs, vegetables, ornamental flowers and foliage, and legumes, including berry plants such as strawberries, blueberries and raspberries, soybeans, potatoes, spinach, cauliflower, tomatoes, tobacco, beans, beets, cotton, peas, squash, melons, canola and the like. As recognized by those skilled in the art, it is necessary to apply preemergence herbicides after the emergence or rooting of the desirable plants, but prior to weed emergence. The precise time of application will vary, depending on the specific crop production system, the area of the country in which the hydrolysate or peptide is applied and the weed species involved. For example, in general, for areas of the upper Midwest, application must be prior to May 1st of any growing season, for control of crabgrass. The invention will be further described by reference to the following detailed examples. EXAMPLE 1 Prior to filtration, liquid com gluten was adjusted to about 14%> solids with water and to pH 6.5 with dilute sodium hydroxide to yield 500 ml of the pH adjusted gluten. Next, 0.07 ml THERMOLASE enzyme (an amylase available from Enzyme Development Corporation, New York, NY) was added. The slurry was jet-cooked while adding steam at 160°C. for 3-4 minutes. To ensure complete liquification of the starch, 0.5 ml of CANALPHA 600 (an amylase from Biocon U.S., Inc., Lexington, Ky.) was added and the slurry held at 80°C. for one hour. The gluten slurry was then cooled to 60°C, and its pH was adjusted to 4.6 with dilute hydrochloric acid. Another enzyme, ZYMETEC 200, 0.2 ml (a glucoamylase manufactured by Enzyme Technology, Inc., Ashland, Ohio), was added to the slurry and the slurry maintained at 60°C. for 13 hours. The slurry was filtered through diatomaceous earth and the filter cake was washed with water. The filtrate and washings were discarded, the wet filter cake reslurried in water to about 12% solids and adjusted to pH 8.5 with Ca(OH) 2 . Then 0.2 ml of the protease enzyme, ALCALASE 2.4L (NOVO Laboratories, Danbury, Conn.) was added while rnaintaining the reaction mixture at pH 8.5, 55°C. for 5 to 8 hours (or until such time that the pH remained constant). Afterwards, dilute phosphoric acid was added to adjust the pH to 6.5 to precipitate the calcium ion as calcium phosphate. The resulting suspension was then heated to 85°C. for 20 minutes to inactivate the enzyme. The solution was filtered and the cake washed with water, followed by combining the washings with the filtrate. The filter cake was discarded. The clear, brown filtrate containing com gluten hydrolysate can be spray dried as is, or reduced by evaporation and then spray dried. The resulting dry product, com gluten hydrolysate, has the properties listed in Table II below. TABLE π Appearance Cream-tan powder Dry substance, %> >90 Solids recovery >50 Protein, % DB (% Kjeldahl nitrogen x 6.25) >90 pH (as 5% solution) >6.5 Water solubility (as 10%) w/v solution) soluble with slight haze Ash, % DB <5 Odor characteristic odor EXAMPLE 2 Com gluten hydrolysate was prepared by a simplified procedure which also yields a water-soluble form of lower protein content. As in Example 1, the liquid com gluten is reconstituted in water, this time to about 10% solids. The slurry (500 ml) was then adjusted to pH 8.5 with a 10% slurry of calcium hydroxide. The protease enzyme, ALCALASE 2.4L (1.0% dry basis), was added and the solution stirred at 60°C. for 5 to 8 hours, or until such time that the pH remained constant at 8.5. The material was then processed as described in Example 1, to yield a com gluten hydrolysate which had the properties shown in Table III, below: TABLE ffl Appearance Cream-tan powder Dry substance, % >90 Solids recovery >50 Protein, % DB (% Kjeldahl nitrogen x 6.25) >70 pH 6.5 Water solubility (10% solution) soluble Ash, % DB <5 Odor characteristic odor EXAMPLE 3 The procedure of Example 2 was further simplified to yield a solubilized form of com gluten of somewhat lower protein content by simply following the steps of Example 2, with the exception that the final filtration step was not carried out. After adjustment to a pH of 6.5 with phosphoric acid, the slurry was freeze-dried. The properties of the resulting product are shown on Table IV below: TABLE IV Appearance Cream-tan powder Dry substance, % >90 Solids recovery % >95 Protein, % DB (% Kjeldahl nitrogen x 6.25) >50 Water solubility (10%> soln) >50% of solids pH 6.5 EXAMPLE 4 A study was conducted in a controlled environment to investigate the effect of the com gluten hydrolysate of Example 1 on αeeping bentgrass and crabgrass. An aqueous dilution of the com gluten hydrolysate of Example 1 in 7 ml watα was applied to blottα papa measuring 42.3 cm 2 at levels of 0 g/dm 2 , 0.12 g/dm 2 , 0.24 g/dm:, 0.36 g/dm 2 , and 0.48 g/dm 2 . Eighteen seeds wαe placed on the blottα papers which wαe then put into petri dishes, and placed into a controlled environmental chambα. The chambα was set at a 16 hr photoperiod and maintained at a constant 25°C. Table V illustrates the pαcentage of germination of the αeeping bentgrass and crabgrass with varying application levels of com gluten hydrolysate of Example 1. TABLE V PERCENTAGE OF GERMINATION OF CREEPING BENTGRASS AND CRABGRASS TREATED WITH CORN GLUTEN HYDROLYSATE Level of Hydrolysate (% germination (g/dm 2 ) Bentgrass Crabgrass 0.00 61 67 0.12 44 6 0.24 11 0 0.36 0 0 0.48 0 0 As can be seen from Table V, com gluten hydrolysate completely stopped germination of αeeping bentgrass at application levels above 0.24 g/dm 2 , and completely stopped germination of crabgrass at application levels above 0.12 g/dm 2 . EXAMPLE 5 A study was conducted comparing the effect of com gluten hydrolysates of Examples 1 and 3 on crabgrass in a greenhouse. The crabgrass was seeded at a rate of 0.19 g/dm 2 onto 58 cm 2 pots filled with a clay loam soil. The hydrolysates of Examples 1 and 3 wαe applied to the surface of the pots at levels of 0, 0.86, 1.72, 3.44, and 6.88 g/dm 2 . The pots wαe then placed on a mist bench for 6 days. Aftα seed genriination, if any, the pots wαe moved to a greenhouse bench and maintained for 15 days. Data wαe collected on the numbα of live shoots from each pot. The study was repeated three times. Table VI illustrates the results of this study. TABLE VI THE EFFECT OF TWO CORN GLUTEN HYDROLYSATES ON THE ESTABLISHMENT OF CRABGRASS SEEDLING ON SOIL IN THE GREENHOUSE Level of Crabgrass Hydrolysate (% of live plants/pot) Hydrol. Hydrol. (g dm 2 ) (Ex. 1) (Ex. 3) 0.00 95 95 0.86 23 57 1.72 4 21 3.44 0 2 6.88 0 0 As can be seen from Table VI, the com gluten hydrolysate of Example 1 reduced the establishment of crabgrass by 76%), 96%, 100%), and again by 100%ι at application levels of 0.86 g/dm 2 , 1.72 g/dm 2 , 3.44 g dm 2 , and 6.88 g/dm 2 , respectively. The com gluten hydrolysate of Example 3 reduced the establishment of crabgrass by 40%, 78%, 98%, and 100% at the same application levels. Thus, while the com gluten hydrolysate of Example 3 is somewhat less effective than the com gluten hydrolysate of Example 1, it is still highly active. EXAMPLE 6 In a furthα study, a comparison was made regarding the effects of com gluten meal and the com gluten hydrolysate of Example 1 on the establishment of pααinial ryegrass (Lolium perenne). Application levels of the dry hydrolysate to the surface of soil pots seeded with L. perenne ranged from 0 to 7.8 g/dm 2 . The pots wαe allowed to stay on the mist bench for a 24-hour period in ordα to moisten the soil without leaching of the water soluble com gluten hydrolysate. Table VII provides the results of this study. TABLE Vπ THE EFFECTS OF CORN GLUTEN MEAL (CGM) AND CORN GLUTEN HYDROLYSATE (CGH) ON THE ESTABLISHMENT OF PERENNIAL RYEGRASS Application level of CGM and CGMH (% inhibition) (g/dm 2 ) CGM CGH 0.00 0 0 1.3 0 0 2.6 0 60 3.9 0 87 5.2 3 97 6.5 0 100 7.8 10 97 The above Table demonstrates the inαeased effectiveness of the com gluten hydrolysate of Example 1 as compared with com gluten meal. Treatment with 5.2 g dm 2 of the com gluten hydrolysate of Example 1 resulted in 97% control. The same level of com gluten meal, howevα, resulted in only 3% control. EXAMPLE 7. ACBD HYDROLYSIS OF CORN GLUTEN MEAL Com gluten meal, 20 g, was placed in a 500 ml round bottomed flask, 230 ml of 2N phosphoric acid added and the mixture was refluxed for 20 hours. The dark sluπy was centrifuged and the dark supematant filtαed. The clear brown filtrate (pH 1) was adjusted to pH 6.5 with 31.5 g calcium hydroxide. The precipitate of calcium phosphate was removed by centrifugation and the clear dark supematant freeze-dried to yield a tan powdα containing 91%) solids, 68.4% protein, as is, and 6.06%> ash, as is. This product was designated acid hydrolysate 1 (AH-1). EXAMPLE 8. ACTO HYDROLYSIS OF THE ENZYME HYDROLYSATE OF CORN GLUTEN Fifteen grams of com gluten hydrolysate (prepared as described in Example 1), was placed in a 500 ml round bottom flask and 160 ml of 2N phosphoric acid added. The solution was refluxed 20 hours, filtαed with the aid of filteraid, and the clear brown filtrate, pH 1, adjusted to pH 7.5 with 30 grams of calcium hydroxide. The precipitated calcium phosphate was removed by filtration and the clear filtrate freeze-dried to yield a tan powdα containing 96.5% dry solids, 82.4% protein, as is, and 7.3%) ash, as is. This product was designated acid hydrolysate 2 (AH-2). EXAMPLE 9. HERBICIDAL ACTIVITY OF AOD HYDROLYSATES The above two products, AH-1 and AH-2, along with com gluten hydrolysates, CGH, prepared as described in Example 1, wαe assayed for their h bicide activity by the following standard laboratory assay procedure. Dilutions wαe prepared in watα of AH-1, AH-2, and CGH to contain 1, 2 and 4 mg./ml. A Whatman No. 1 filtα papa disk (7 cm in diameta) was placed in each of several 100 x 15 mm plastic petri dishes. One ml of each of the hydrolysate dilutions was then distributed uniformly onto the filtα papa disks. Thai tai pa inial ryegrass seeds wαe distributed uniformly on top of each filtα pap disk. The petri dishes wae covaed, sealed with parafilm, and allowed to stand at about 23°C. for 14 days. A control sample was prepared in the same mannα except one ml of watα was used. Aftα 14 days, the length of the individual roots of each seed wαe determined and the average of the seven longest roots calculated. This value was expressed as a pαcent of the average root length of the Control. These results are shown in Table VIII, below. TABLE Vffl THE EFFECTS OF ACID HYDROLYSATES OF CORN GLUTEN MEAL (AH-1) AND CORN GLUTEN HYDROLYSATE (AH-2) AND CGH ON THE ESTABLISHMENT OF PERENNIAL RYEGRASS Application level (% of root control length) (mg dm 2 ) AH-1 AH-2 CGH 2.6 68 50 42 5.2 0 0 0 10.4 0 0 0 As the results show, CGH appears to be slightly more effective in inhibiting root formation than either AH-1 or AH-2 but all hydrolysates completely inhibited root growth at 5.2 mg/dm 2 and above. These results furfhα demonstrate that acid treatment can also be used to solubilize the hαbicide activity in plant proteins. Those skilled in the art would realize that othα acids (i.e., hydrochloric, sulfuric, etc.) undα the appropriate conditions could be employed, as well as phosphoric acid, to solubilize the hαbicide activity in plant proteins. EXAMPLE 10 Soy hydrolysate and extracts obtained by the fractionation of soy hydrolysate with an absorptive resin, AMBERLITE XAD-16 (manufactured by Rohm & Haas Company, Philadelphia, Pa.) have also been shown to have hαbicidal activity. In this example, a soy hydrolysate was prepared by slurrying 100 grams of soy protein in about 900 ml of watα (adjusted to a pH 5.5 with hydrochloric acid). About 0.5 grams of RHOZYME-54 (a protease manufactured by Rohm & Haas Company, Philadelphia, Pa.) was then added. The slurry was maintained at 45°-50°C. for 5 hours, and then heated to 90°C. for 10 minutes to inactivate the enzyme. The slurry was then centrifuged. The hazy effluent was treated with 1-2% activated carbon (dry basis) at 60°C for about 30 minutes and then filtαed with the aid of diatomaceous earth, producing a clear brown filtrate of soy hydrolysate. Seventy-five ml of the soy hydrolysate containing 7.16 grams of dissolved fluids wαe passed through a 2.54 cm x 35.6 cm column of Ambαlite XAD-16 resin (185 ml bed volume) at 3-5 ml pα minute, followed by a watα wash. When the solids content of the effluent had dropped to zαo, as determined by its refractive index, the collected effluent (360 ml, colorless, slightly hazy, pH 5.6) was freeze-dried to produce a white powdα (Fraction 1). The column was then washed with 88%> methanol until the effluent showed no solids. The brown colored effluent was evaporated to remove the alcohol and the aqueous concentrate was freeze dried to yield a tan/brown powdα (Fraction 2). EXAMPLE 11 The com gluten hydrolysate of Example 1, the soy hydrolysate of Example 10, and the fractions derived from soy hydrolysate as described in Example 10 wαe tested for their ability to inhibit root formation of pαennial rye-grass. One ml of aqueous dilutions of the samples was applied to 7 cm diametα Whatman No. 1 filtα papa held in 100 x 15 mm petri dishes. Ten paennial ryegrass seeds wαe added, the dishes covαed, sealed with parafilm and held for 16 hours at 25°C. with continuous lighting. The papers wαe then held at 11°C. in the dark for 8 hours. This lighting-tempαature cycle was repeated for 14 days. Root length was then determined and expressed as a pαcentage of the untreated control. The results are shown on Table IX below. TABLE IX THE EFFECT OF CORN GLUTEN HYDROLYSATE OF EXAMPLE 1 (CGH-1) SOY HYDROLYSATE, AND FRAC¬ TIONS FROM SOY HYDROLYSATE (SH) OF EXAMPLE 10 ON THE ESTABLISHMENT OF PERENNIAL RYEGRASS Application Average Level Root Length Pαcent Sample (mg/dm 2 ) (mm) of Control Control 0 52.3 100 CHG-1 2.6 25.3 48 SH 2.6 43.4 83 SH-Fraction 1 2.6 14.0 27 SH-Fraction 2 2.6 19.4 37 As can be seen from Table IX above, soy hydrolysate was not as effective at preventing the establishment of pααinial ryegrass as was the com gluten hydrolysate of Example 1. The soy hydrolysate Fractions 1 and 2, howevα, wαe more effective in inhibiting root formation of pαennial ryegrass than was the com gluten meal hydrolysate of Example 1. EXAMPLE 12. ENZYME HYDROLYSIS OF SOY PROTEIN Fifty grams of PROFAM 90 (soy protein isolate manufactured by Grain Processing Corporation, Muscatine, Iowa) was slurried in 700 ml of watα. The thick slurry was adjusted from pH 7.3 to pH 8.5 with 10% Ca(OH) 2 . ALCALASE 2.4L (0.5 ml) was added and the slurry was continuously stiπed at 55°C. The pH was maintained between pH 8 to 8.5 with Ca(OH) 2 . When the pH had remained constant, the pH was adjusted to pH 6.5 with 10% v/v H 3 P0 4 to precipitate calcium as Ca 3 (P0 4 ) 2 . Aftα heating the slurry in a steam chest for 20 minutes to inactivate the enzyme, it was filtαed with filteraid and the residue discarded. The clear tan filtrate was freeze dried to a tan powdα. The product contained 94.7% dry solids and 91.7%) protein, as is, and was designated soy hydrolysate 1 (SH-1). EXAMPLE 13. ENZYME HYDROLYSES OF WHEAT GLUTEN Fifty grams of wheat gluten (Sigma Chemical Co., St. Louis, Mo.) wαe slurried in 500 ml of watα and treated as described in Example 12. The resulting hydrolysate product was freeze dried to a tan powdα containing 98.1%> dry solids and 88.8% protein, as is, and was designated wheat hydrolysate, WH-1. EXAMPLE 14 The above products of Examples 12-13, SH-1 and WH-1, along with com gluten hydrolysate, CGH, prepared as described in Example 1, wαe assayed for their hαbicide activity by the in vitro assay as described in Example 9. For the assay, dilutions of SH-1, WH-1, and CGH wαe prepared to contain 1.3, 2.6, 3.9 and 5.2 mg/dm 2 . The results of the assay after 14 days are show in Table X TABLE X The Effects of Enzyme Hydrolysates of Com Gluten (CGH), Soy Protein (SH-1), and Wheat Gluten (WH-1) on the Establishment of Pααinial Ryegrass Application level (% of Control Root Length) (mg / dm 2 ) CGH SH-1 WH-1 0.0 100 100 100 1.3 100 90 69 2.6 21 43 47 3.9 0.3 0.6 18 5.2 00 0.9 1.8 As the data in Table X demonstrate, all protein hydrolysates contain significant levels of hαbicide activity, especially at the 2.6 mg/dm 2 level and above. CGH appears to be the most effective, followed closely by SH-1, with WH-1 last. It is believed that othα grain and plant proteins comprise hαbicidal activity which could be isolated and concentrated in a similar fashion. EXAMPLE 15. ISOLATION AND OIARACTEREATΪON OF BIOACIΓVΈ DIPEPTIDES The bioactive dipeptides wαe isolated and characterized employing the following procedures. (1) Column Chromatography: an aqueous solution of the com gluten hydrolysate of Example 1 (10%> solids) was loaded on a Sephadex G- 15 resin (Pharmacia) gel filtration column (28 x 998 mm)(αoss-linked dextran, exclusion limit < 1,500 daltons). Bioactive fractions of the eluate wαe identified with bioassays, then pooled and subjected to furthα purification steps. The bioassays wαe conducted in petri dishes using 10 pαennial ryegrass (Lolium pererme) seeds placed on 1 layα of Whatman #1 filtα papa of 38.5 cm 2 in area. The dishes wαe sealed with parafilm and placed in a controlled environmental chamber. The light intensity in the growth chambα was 70 μmol/m 2 s at 25°C./15°C. day/night temperature with a 16 hr photoperiod. One ml of a given eluate fraction was applied to the filtα papa in each petri dish. The study was conducted with seven replications for each eluate fraction. (2) The bioactive fractions identified in step 1 wαe injected into a high performance liquid chromatograph (HPLC) equipped with a reverse phase (RP) C18 column (DYNA-MAX), 5μ, 10.0 x250 mm) using a methanol in watα gradiαit (0-5%> methanol) for 10 minutes with a flow rate of 4 ml/min. The bioactive peak was isolated and subjected to amino acid analysis and peptide sequencing. (3) Purified samples obtained from step 2 wαe dαivatized with phenylisothiocyanate (PITC) to form phenylthiocarbamyl (PTC) peptides which wαe resolved using HPLC equipped with a naπow-bore (2.1 x 250 mm) C18-RP column (VYDAC) using 5% to 45% B in A in 35 min (A=0.1% TFA in H 2 0; B=0.08% trifluoroacetic acid in CH 3 CN) at a flow rate of 300 μl/min. (4) The polypeptides wαe sequenced on a Biosystem 477A Protein Sequencα with a 120A PTH Amino Acid Analyzα. The isolated bioactive peak was resolved into 5 dipeptides: Gln-Gln, Ala-Asn, Ala-Gin, Gly-Ala and Ala-Ala. EXAMPLE 16. HERBICIDAL ACTIVITY OF S YNTHEIΪC DIPEPTIDES Four synthetic dipeptides of the same structure as the dipeptides identified from the sample purified from the com gluten hydrolysate wαe obtained from the Sigma Chemical Co., St. Louis, Mo. The fifth, Gln-Gln, was obtained from Bachem Bioscience Inc. The activity of each synthetically derived pφtide was tested in seven rφlications on pααinial ryegrass using the same bioassay technique described in Example 9 in the presence of inαeasing amounts of the dipφtides mixed with distilled watα (Table XI). TABLE XI Root-Inhibiting Activity of the Five Identified Dipφtides on Pαennial Ryegrass Seeds. Root Length of Pααinial Ryegrass Seedlings Expressed as a Pαcentage of the Control (%), Average of 2 Trials Dipφtides μg/cm 2 * Gln-Gln Ala-Asn Ala-Gin Gly-Ala Ala-Ala 0 100 100 100 100 100 8 107 101 83** 95 74** 13 89 85 70 68 44 21 82 63 49 29 27** 26 63 29 41 16 5 31 12 2 4 9 I** 39 3* 0 0 2 2 52 0 0 0 0 0 *Each dish contained a Whatman No. 1 filtα measuring 38.5 cm 2 in area. **Only one trial was performed. All of the dipφtides excφt Gln-Gln reduced rooting of the pαennial ryegrass at least 50%) at the 26 μg/cm 2 rate. Thαe was almost total inhibition of rooting at highα rates. The most effective dipφtides wαe Ala-Asn and Ala- Ala. EXAMPLE 17 In a study designed to verify the bioactivity of the dipφtides on soil, Ala-Gin and Gly-Ala wαe applied to the surface of 56.3 cm 2 plastic pots filled with a Nicollet (fine, loamy, mixed mesic, Aquic Hapludol) soil. The two dipφtides wαe applied at 0 to 3552 mgtøm 2 . Data wαe collected 21 days aftα treatment on pαcentage survival of seedlings and on the mean length of roots in mm. The test specie was αeφing bentgrass (Agmstis pdυstήs). The results are shown on Table XII below. TABLE Xπ PERCENTAGE (%) OF CREEPING BENTGRASS PLANTS SURVIVING 25 DAYS AFTER TREATMENT, AND MEAN ROOT LENGTH OF SEEDLINGS (mm) Dipφtide Ala-Gin Glv-Ala (mg/dm 2 ) -%- ■mm- -%- ■ mm- 0 100 25 100 25 89 100 20 105 20 178 40 15 40 15 355 30 2 30 5 710 25 0 25 2 1066 5 0 25 2 1776 0 0 0 0 3552 0 0 0 0 The data in Table XII indicate that Ala-Gin and Gly-Ala can completely inhibit the emαgαice and establishment of αeφing bentgrass at concentrations of 1.8 g/dm 2 and above, and are partially effective at much lowα concentrations. EXAMPLE 18. PENTAPEPTIDE The active compound was isolated from com gluten hydrolysate by the following procedure: (1) Column Chromatography. An aqueous solution of gluten hydrolysate (10%) was loaded onto a Sφhadex G-15 resign (Phamacia) gel filtration column (28X998mm). Bioactive fractions, which wαe idαitified with the bioassay as described in Example 15, wαe subjected to furthα purification stφs. (2) High Performance Liquid Chromatography (HPLC) on C18 reversed phase semiprφ column. Bioactive fractions prφared from stφ (1) wαe injected onto HPLC reversed phase (RP) C18 column (Waters, 5μ, 300X7.8 mm I.D.) using a methanol in watα gradiant (0 - 25%> methanol) in 50 minutes with a flow rate of 4mI7min. Five peaks wαe collected having retention times (Rt) between 20 and 30 minutes. The activity of the fractions pooled from 18 runs was tested on pααinial ryegrass in petri dish using 38.5 cm 2 Whatman #1 filtα papa grown unda controlled environmental conditions with fluorescent lighting (70 μmol/mV) at 25°C for 16hr, and in the dark at 15°C for 8hr. Seedlings wαe measured and, based on their root length and shoot length, compared to the mean of the measurements of germinated seeds on the control, which had lmL distilled deionized watα (D.D. H 2 0) in each plate (Table XIII). From bioassay results, peak #4 with Rt=25.5 was found to be bioactive. TABLE Xffl Bioactivity of HPLC Frances of Hydrolysate Pααinial Ryegrass Petri Dish Bioassay * HPLC Fraction Pαcentage of Control Root Length * Y%) # Root Length 1 104.5+5 2 103.4±8.6 3 93.2+25.1 4 51.5+5.9 5 102.3+40.0 * Each dish contained a Whatman No. 1 filtα pap measuring 38.5 cm 2 in area. "Control had lmL D.D H 2 0/plate and was considαed as 100%. Values are means of measurement of the length of 7 seedlings ± standard deviation. (3) High Performance Liquid Chromatography (HPLC) on a naπow-bore RP C-18 column. The isolated bioactive peak from stφ (2) was subjected to a furthα purification using a naπow-bore RP C-18 HPLC column (Vydac, 250X2.1 mm I.D.) with a acetonitrile in watα (5 - 25% acetonitrile) linear gradiant ova 40 minutes with a flow rate of 200μL/min. A major peak was isolated subjected to pφtide sequencing on a Biosystems 477A Protein Sequencα with a 120A PTH Amino Acid Analyzα and Lasα Desorption Mass Spectrometry (Finnigan, LASERMAT) and identified as Leu-Sα-Pro- Ala-Gin. The compound was synthesized by the Protein Facility at Iowa State University and its bioactivity was tested on the pαennial ryegrass bioassay. Seedlings wαe measured for their root length and shoot length and compared against the control (Table XTV). TABLE XIV Bioactivity of Synthetic Pφtide Pαennial Ryegrass Petri Dish Bioassay * Pφtide T^u-Sα-Pro-Ala-Gln. MW=514.6 μg / cm 2 Pαcentage of Control * Y%) Root Length Shoot Length 13 50.1+16.9 14.5+3.2 26 44.2±17.6 15.5+2.7 39 27.7+7.1 11.6+2.7 52 10.2+9.1 13.5+4.4 104 0 0.5+1.3 * Each dish contained a Whatman No. 1 filtα papa measuring 38.5 cm 2 in area. Only one trail was performed. "Control had lmL D.D H 2 0/plate and was considαed as 100%. Values are means of measurement of the length of 7 seedlings ± standard deviation. The pentapφtide showed inhibitory activity to both root and shoot growth of germinating ryegrass. Its root-inhibiting activity was as potent as alaninyl-alanine (ala-Ala), the most active dipφtide isolated from com gluten hydrolysates at the rate of 13 μg/cm 2 . It could inhibit up to 90%> root growth at the rate of 52 μgføn 2 . Howevα, the molecular weight is 514.6 for the pentapφtide verses to 160.2 for Ala-Ala, thαefore, the bioactivity of the pentapφtide is highα than that of the dipφtides on a molar basis. All publications, patents and patent documents are incorporated by refαence hαein, as though individually incorporated by refααice. The invention has been described with refααice to various specific and prefeπed embodiments and techniques. Howevα, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims: WHAT IS CLAIMED IS: 1. A method for inhibiting growth of undesirable plants in a plot of soil comprising applying an amount of a plant protein hydrolysate to the plot of soil, prior to emαgαice of the unwanted plants, whαein said amount inhibits the growth of the undesirable plants by inhibiting their root development. 2. The method of claim 1 whαein the plant protein hydrolysate is selected from the group consisting of com gluten hydrolysate, wheat gluten hydrolysate, soy protein hydrolysate and mixtures thαeof. 3. The method of claim 1 whαein the plot of soil also comprises desirable plants. 4. The method of claim 3 whαein the plant protein hydrolysate is applied aftα the emαgαice of the desirable plants. 5. The method of claim 1 furthα comprising transplanting desirable plants into the plot of soil. 6. The method of claim 1 whαein the plant protein hydrolysate is applied at a concentration of about 0.5 ^dm 2 to 10 g/dm 2 of soil. 7. The method of claim 2 whαein the plant protein hydrolysate is com gluten hydrolysate. 8. The method of claim 7 whαein the com gluten hydrolysate has a protein content (dry basis) of at least about 50%>. 9. The method of claim 8 whαein the com gluten hydrolysate has a protein content of about 90%. 10. The method of claim 1 whαein the undesirable plants are broadleaf weeds. 11. The method of claim 1 whαein the undesirable plants are grassy weeds. 12. The method of claim 1 whαein the desirable plants are already established and are monocotyledonous. 13. The method of claim 11 whαein the desirable plants are turfgrasses. 14. The method of claim 1 whαein the desirable plants are dicotyledonous plants. 15. The method of claim 1 wherein the desirable plants are berry plants. 16. The method of claim 15 whαein the desirable plants are strawberries. 17. The method of claim 1 wherein the plant protein hydrolysate is dissolved or suspended in watα and applied to the plot of soil by spraying or watering. 18. The method of claim 1 whαein the amount of plant protein hydrolysate is mixed with the soil. 19. An hαbicidal compound comprising an herbicidally effective amount of a plant protein hydrolysate in combination with a compatible carriα vehicle. 20. The method of claim 19 whαein the plant protein hydrolysate is selected from the group consisting of com gluten hydrolysate, wheat gluten hydrolysate, soy protein hydrolysate and mixtures thαeof. 21. The composition of claim 19 wherein the carriα vehicle is a liquid vehicle. 22. The composition of claim 21 whαein the liquid vehicle is watα. 23. The composition of claim 19 wherein the carriα vehicle is a solid. 24. A method for selectively inhibiting the growth of undesirable plants in a plot of soil comprising applying an amount of a pφtide selected from the group consisting of Gln-Gln, Ala-Asn, Ala-Gin, Gly-Ala, Ala-Ala, Leu-Sα- Pro-Ala-Gln, and mixtures thαeof, to the plot of soil prior to the emαgence of the undesirable plants, wherein said amount is effective to inhibit the growth of the undesirable plants by inhibiting their root development. 25. The method of claim 24 wherein the plot of soil also comprises desirable plants and the pφtide is applied aftα the emαgence of the desirable plants, at a level which does not inhibit the growth of the desirable plants. 26. The method of claim 24 fia hα comprising planting desirable established plants in the plot of soil, which desirable plants are not inhibited in their growth by the amount of the pφtide applied to said plot of soil. 27. The method of claim 26 furthα comprising applying an additional amount of said pφtide to said plot of soil, so that the emαgence of undesirable plants is inhibited while not inhibiting the growth of said desirable plants. 28. The method of claims 24, 25, or 26 whαein the pφtide is applied in combination with a liquid carriα vehicle. 29. The method of claim 28 whαein the pφtide is applied as an aqueous solution or dispersion. 30. The method of claims 24, 25, or 26 whαein the pφtide is applied in combination with a solid carriα vehicle. 31. The method of claims 24, 25, or 26 whαein the pφtide is applied to the soil plot at a concentration of about 0.25-5 g/dm 2 . 32. The method of claims 24, 25, or 26 wherein the undesirable plants are grassy weeds. 33. The method of claims 24, 25, or 26 whαein the undesirable plants are broadleaf weeds. 34. The method of claims 24, 25, or 26 whαein the undesirable plants are monocotyledonous. 35. The method of claim 34 wherein the desirable plants are turfgrasses. 36. The method of claims 25 or 26 wherein the desirable plants are dicotyledonous plants. 37. The method of claim 36 wherein the desirable plants are berry plants or ornamental flowers. 38. The method of claim 36 wherein the desirable plants are strawberry plants. 39. A preemαgαice hαbicidal composition comprising an hαbicidally effective amount of a pφtide selected from the group consisting of Gln-Gln, Ala-Asn, Ala-Gin, Gly-Ala, Ala-Ala, Leu-Sα-Pro-Ala-Gln, and mixtures thαeof, in combination with a compatible carriα vehicle. 40. The composition of claim 39 whαein the carriα vehicle is a liquid. 41. The composition of claim 40 whαein the composition is an aqueous solution or dispersion of said pφtide. 42. The composition of claim 39 whαein the carriα vehicle is a finely divided solid.

Inventors: CHRISTIANS NICK E, GARBUTT JOHN T, LIU DIANNA, CHRISTIANS, NICK, E., GARBUTT, JOHN, T., LIU, DIANNA
Applicants: GRAIN PROCESSING CORP, UNIV IOWA RES FOUND, GRAIN PROCESSING CORPORATION, IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC.
Application year: 1994
Main classification: A01N37
IPC codes: A01N37, A01N43, A01N61, A01N65